Department of Psychiatry, University of British Columbia, Vancouver, BC, V6T 2A1, Canada. simpson@cmmt.ubc.ca.

Abstract

BACKGROUND:

Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters-however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo.

METHODS:

For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were "cut down" to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP.

RESULTS:

The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia.

CONCLUSIONS:

Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.

“Plug and Play” rAAV2 genome plasmid used to clone MiniPromoters (MiniPs) upstream of icre or EmGFP enables high throughput pipeline testing. a Plasmids were generated containing either the icre or the EmGFP reporter. An AsiSI site flanks a removable WPRE. MiniPs were cloned at the MCS using a combination of the four available cut sites. The plasmids are subsequently used to generate single stranded rAAV. b Screening step one, an historical-indirect reporter system. MiniPs drive expression of icre, which in turn recombines the endogenous loxP sites and removes the stop sequence 5ʹ of the lacZ gene, thus driving expression of β-galactosidase from the strong ubiquitous ROSA26 promoter. c Screening step two, a direct reporter system. MiniPs drive direct expression of EmGFP, which can be imaged by epifluorescence or signal amplified using antibodies. bp, base pairs; ITR, inverted terminal repeat; MCS, multiple cloning site; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element